A New Concept of Enhancing Immuno-Chemotherapeutic Effects Against B16F10 Tumor via Systemic Administration by Taking Advantages of the Limitation of EPR Effect

Abstract

The enhanced permeability and retention (EPR) effect has been comfortably accepted, and extensively assumed as a keystone in the research on tumor-targeted drug delivery system. Due to the unsatisfied tumor-targeting efficiency of EPR effect being one conspicuous drawback, nanocarriers that merely relying on EPR effect are difficult to access the tumor tissue and consequently trigger efficient tumor therapy in clinic. In the present contribution, we break up the shackles of EPR effect on nanocarriers thanks to their universal distribution characteristic. We successfully design a paclitaxel (PTX) and alpha-galactosylceramide (αGC) co-loaded TH peptide (AGYLLGHINLHHLAHL(Aib)HHIL-Cys) -modified liposome (PTX/αGC-TH-Lip) and introduce a new concept of immuno-chemotherapy combination via accumulation of these liposomes at both spleen and tumor sites naturally and simultaneously. The PTX-initiated cytotoxicity attacks tumor cells at tumor sites, meanwhile, the αGC-triggered antitumor immune response emerges at spleen tissue. Different to the case that liposomes are loaded with sole drug, in this concept two therapeutic processes effectively reinforce each other, thereby elevating the tumor therapy efficiency significantly. The data demonstrates that the PTX/αGC-TH-Lip not only possess therapeutic effect against highly malignant B16F10 melanoma tumor, but also adjust the in vivo immune status and induce a more remarkable systemic antitumor immunity that could further suppress the growth of tumor at distant site. This work exhibits the capability of the PTX/αGC-TH-Lip in improving immune-chemotherapy against tumor after systemic administration.

Keywords: Alpha-galactosylceramide; Cancer immune-chemotherapy.; Distribution characteristic; Liposome; Paclitaxel.

Scheme 1

Diagram of PTX/αGC-TH-Lip mediated combination of chemotherapy and immunotherapy against tumor. The intravenously injected PTX/αGC-TH-Lip will function at two major sites during circulating in vivo: A. A relatively small part of the liposomes will accumulate at tumor sites (primary tumor), with PTX killing the tumor cells directly. B. A large proportion of the liposomes will finally settle at the spleen and liver, where the αGC would be presented on the CD1d molecules in APCs and further activate the iNKT cells, leading to the ongoing production of large amounts of IFN-γ. C. The IFN-γ could subsequently activate other cell types, such as NK cells and macrophages in the innate immune system as well as CD8⁺ T cells in the acquired immune system. D. The PTX-induced tumor cell death in tumor sites could boost the tumor-associated antigens release and the tumor antigen presentation process is stimulated by IFN-γ secreted from iNKT cells, bringing about an amplified tumor-specific CD8⁺ T cells cytolytic effect. E. The activated NK cells, macrophages and CD8⁺ T cells would contribute a lot to eliminating the circulating tumor cells and ease metastasis status. F. The tumor-specific CD8⁺ T cells enter the systemic circulation and will be recruited to tumors at distant sites to trigger the “effector phase” of the adaptive immune response.

Figure 1

The characterization of PTX/αGC-TH-Lip. (A) The variations in turbidity (represented by transmittance) of PTX/αGC-TH-Lip, PTX-TH-Lip, TH-Lip and PEG-Lip in 50% FBS (n=3, mean ± SD). (B) The PTX release profiles of free PTX, PTX/αGC-TH-Lip and PTX-TH-Lip in PBS over 48 h under different pH conditions (n=3, mean ± SD).

Figure 2

The accumulation of liposomes in organs and tumors. (A) Ex vivo analysis of organs from B16F10 tumor bearing mice by NIR fluorescence imaging at 1 h, 4 h and 24 h post-injection of DiD-loaded TH-Lip (upper) or PEG-Lip (lower). (B) NIR fluorescence imaging of spleens and tumors from B16F10 tumor bearing mice at 1 h, 4 h and 24 h post-injection of DiD-loaded TH-Lip (upper) or PEG-Lip (lower), and the comparison of the fluorescence intensity in spleen and tumor at 24 h between DiD-loaded TH-Lip and PEG-Lip. (C) The fold increase of fluorescence intensity in liver, spleen and tumor (the fluorescence intensity of each tissue at 4 h or 24 h / that at 1 h). Values are the mean ± SD (n=3).

Figure 3

The secretion of IFN-γ after intravenous administration of αGC formulations or free αGC. (A) The production of IFN-γ after intravenous administration of αGC-loaded liposomes with the concentration of αGC ranging from 0 to 25 μg/mL. (B) The production of IFN-γ after primary and secondary administration of liposome-formulated αGC or free αGC (αGC concentration was 25 μg/mL, and the injection volume was 200 μL), the following operation was the same as above. (C) IFN-γ production after application of different PTX/αGC-TH-Lips in which the concentration of PTX was adjusted to 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/mL, respectively (αGC concentration was 25 μg/mL, and the injection volume was 200 μL). (D) IFN-γ production after application of αGC-TH-Lips or αGC-Lips. (n=5, *P < 0.05, **P < 0.01).

Figure 4

αGC presentation on CD1d molecules in antigen presenting cells (APCs). APCs presenting αGC on CD1d were identified as MHC-II⁺αGC/CD1d⁺ cells. (A-E) The typical dot plots of the splenocyte derived from non-treated mouse, free αGC treated mouse αGC-TH-Lip treated mouse, PTX/αGC-Lip treated mouse and PTX/αGC-TH-Lip treated mouse. The numbers in the top right area indicate the percentage of MHC-II⁺ αGC /CD1d⁺ cells. (F) The quantitative analysis of flow cytometry data. Values are the mean ± SD (n=3, *P <0.05).

Figure 5

Cellular uptake of CFPE-labeled TH-Lip and PEG-Lip on DC2.4 cell. (A) The fluorescence peak of TH-Lip and PEG-Lip on flow cytometry histogram. (B) The quantitative result of the fluorescence intensity in DC2.4 cells incubated with TH-Lip or PEG-Lip. Values are the mean ± SD (n=5, **P <0.01). (C) — (D) The confocal images of cellular uptake of TH-Lip and PEG-Lip with different magnification. Scale bars represent 20 μm and 5 μm, respectively.

Figure 6

The expression status of CD86, CD40 and CD80 on CD11c⁺ dendritic cells from spleens. Diagrams are the typical dot plots of the splenocyte derived from non-treated mouse, Hepes treated, free PTX&αGC treated mouse, αGC-TH-Lip treated mouse, PTX-TH-Lip treated mouse and PTX/αGC-TH-Lip treated mouse. The numbers in the top right area indicate the percentage of CD86⁺CD11c⁺cells (A), CD40⁺CD11c⁺cells (B) and CD80⁺CD11c⁺cells (C).

Figure 6

The expression status of CD86, CD40 and CD80 on CD11c⁺ dendritic cells from spleens. Diagrams are the typical dot plots of the splenocyte derived from non-treated mouse, Hepes treated, free PTX&αGC treated mouse, αGC-TH-Lip treated mouse, PTX-TH-Lip treated mouse and PTX/αGC-TH-Lip treated mouse. The numbers in the top right area indicate the percentage of CD86⁺CD11c⁺cells (A), CD40⁺CD11c⁺cells (B) and CD80⁺CD11c⁺cells (C).

Figure 7

Relative populations of immune cells (CD8α⁺ T cells, NK cells) in spleens at Day 7 after twice systemic administrations of samples. (A) The relative amount of CD8α⁺ T cells in spleens. (B) The relative amount of NK cells in spleens.

Figure 8

The anti-tumor assay of different liposome preparations or free drugs on B16F10 tumor bearing C57BL/6 mice. (A) Tumor growth curves of mice receiving different in vivo treatments (n=7, mean ± SD). (B) Photographs of tumors at the end of treatment. (C) HE staining of tumor tissues after treatment. a: Hepes; b: αGC-TH-Lip; c: Free drugs; d: PTX -Lip; e: PTX/αGC-TH-Lip. Scale bars represent 50 μm.

Figure 9

(A) The Cytotoxicity Assay of CD8⁺ T lymphocytes against B16F10 cells. B16F10 tumor-bearing mice were given systemic injection of Hepes or different formulations containing 5 μg of αGC at the 10^th day and 15^th day after tumor inoculation. The splenocytes were collected a week later followed by mitomycin C-treated B16F10 tumor cells and mIL-2 (5 U/mL) stimulation in vitro. Cytotoxic activity of the splenocytes was determined by lactate dehydrogenase (LDH) assay. “E/T ratio” represents effector/target cell ratio. (n=3, mean ± SD). (B) Therapeutic effects on experimental lung metastasis model with B16F10 melanoma. The diagram above indicates the experimental scheme. The lung tissues were collected at day 15, day 19 and day 23, respectively. The metastasis statuses are exhibited in the picture below. (C) Growth of the distant (contralateral to the primary tumor) untreated tumor over time. (n=7, mean ± SD).